Abstract Introduction Medulloblastoma is a rapidly growing embryonal-CNS tumor with a 5-year survival-rate of nearly 0% in recurrent-cases. Current non-invasive monitoring relies primarily on imaging, which lacks the necessary sensitivity and fails to capture molecular features associated with tumor progression. Liquid-biopsies have emerged as a less invasive alternative that studies serially collectible sources of tumor-derived biomolecules. Relapsed Group 4 medulloblastomas are particularly challenging due to their poor prognosis and lack-of-molecular-biomarkers to predict relapse. This study aims to establish robust-assays for monitoring tumor progression, recurrence, and treatment response using liquid-biopsies to guide rational therapeutic interventions. Methods We used patient-derived cerebrospinal fluid (CSF; n = 7 patients) and plasma (n = 14 timepoints from n = 5 patients) to analyze longitudinal genetic, and immunological profiles with nanopore-sequencing and Mesoscale-Discovery (MSD) assay. Results Nanopore-sequencing assay detected copy-number-variation (CNVs) and distinct methylation patterns in group 4 medulloblastoma. Optimized protocols yielded high-purity cfDNA (64-97%; 20-376 ng) from just 500µL CSF, enabling consistent detection of tumor-derived-CNVs and methylation profiles, even at shallow-sequencing depths. Comparison against the gold-standard Illumina-EPICarray validated the effectiveness of nanopore-sequencing in generating similar CNV profiles using only 1/10th of cfDNA input. Matched-tumor-tissue vs CSF-CNV analysis highlighted the assay’s sensitivity in capturing further tumor heterogeneity. Additionally, we optimized a multianalyte ELISA (MSD) assay for monitoring treatment response and progression in patients undergoing immunotherapy in various clinical trials. The standardized MSD assay demonstrated a low pg/mL detection-limit and a 7-fold-dynamic detection-range, using only 50µL plasma. Longitudinal-immune- profiling in five medulloblastoma patients demonstrated an expected elevation in proinflammatory cytokines in Group 3/4 patients undergoing immunotherapy. Impact This multi-dimensional approach highlights the utility of such assays for monitoring progression and treatment-response using serial-liquid-biopsies, paving-the-way for precision-oncology in medulloblastoma-care. However, further investigation on a larger cohort will help toestablishstatistical significance. 1. Patel, A. et al. Prospective, multicenter validation of a platform for rapid molecular profiling of central nervous system tumors. Nat. Med. 31, 1567–1577 (2025). 2. Afflerbach, A.-K. et al. Classification of brain tumors by nanopore sequencing of cell-free DNA from cerebrospinal fluid. Clin. Chem. 70, 250–260 (2024). 3. Vermeulen, C. et al. Ultra-fast deep-learned CNS tumour classification during surgery. Nature 2023 622:7984 622, 842–849 (2023). 4. Kuschel, L. P. et al. Robust methylation-based classification of brain tumours using nanopore sequencing. Neuropathol Appl Neurobiol 49, (2023). 5. Liu, A. P. Y. et al. Serial assessment of measurable residual disease in medulloblastoma liquid biopsies. Cancer Cell 39, 1519-1530.e4 (2021). 6. Liu, Y. et al. DNA methylation-calling tools for Oxford Nanopore sequencing: a survey and human epigenome-wide evaluation. Genome Biol 22, 1–33 (2021).
Singh et al. (Tue,) studied this question.